Probes for detection of human papillomavirus

ABSTRACT

The presence of human papillomavirus (HPV) in a sample can be detected and the HPV typed by a method that involves the amplification of HPV DNA sequences by the polymerase chain reaction (PCR). The primers used in the method are consensus primers that can be used to amplify a particular region of the genome of any HPV. The presence of HPV in a sample is indicated by the formation of amplified DNA. The HPV is typed by the use of type-specific DNA probes specific for the amplified region of DNA.

CROSS-REFERENCE

This application is a continuation-in-part of copending U.S. patent application Ser. No. 243,486, filed Sep. 9, 1988, which is incorporated herein by reference.

The present invention provides methods for detecting human papillomavirus (HPV) by the polymerase chain reaction (PCR).

BACKGROUND

1. Field of the Invention

The present invention provides medical diagnostic methods for detecting and typing HPV. The method utilizes PCR, a DNA amplification technique widely used in the fields of molecular biology and genetic engineering. The method can also be used to generate information concerning previously known strains of HPV and consequently has applications in the field of virology

2. Description of Related Disclosures

Papillomaviruses have been linked to widespread, serious human diseases, especially carcinomas of the genital and oral mucosa. And although genital HPV infection is associated with cancer primarily in women, recent evidence suggests that HPV may play a role in the development of prostate cancer in man. Broker et al., 1986, Cancer Cells 4:17-36, review the molecular, cellular, and clinical aspects of the papillomaviruses and the relationship of HPVs to cancer. HPV types 6, 11, 16, 18, and 33 are known HPV types in the human population, and Broker et al., 1986, Cancer Cells 4:589-594, disclose that HPV types 6, 11, and 18 share significant homology at the DNA level, particularly at the L1 open reading frame.

Identification and typing of HPV is quite important, because different types of HPV pose different risks to the affected individuals. For instance, HPV16 and HPV18 have been more consistently identified in higher grades of cervical dysplasia and carcinoma than other HPV types. Webb et al., December 1987, J. Inf. Disease 156(6):912-919, report a method for detecting HPV DNA types that utilizes a reverse-blotting procedure. The procedure involved forming a membrane to which genomic DNA from four different HPV types was bound and then hybridizing labelled DNA from a biological sample to the DNA bound to the membrane. Caussey et al., February 1988, J. Clin. Microbiol. 26(2):236-243 describe similar HPV detection methods.

Shibata et al., January 1988, J. Exp. Med. 167:225-230, disclose the use of PCR to amplify and detect the presence of HPV16 and HPV18 DNA. U.S. Pat. Nos. 4,683,195 and 4,683,202 disclose PCR and the use of PCR to detect the presence or absence of nucleic acid sequence in a sample. European Patent Publication Nos. 229,701 and 269,445 disclose the use of PCR to amplify and detect DNA sequences associated with a wide variety of viruses, including the AIDS virus, HTL V I, and HTL V II.

Maitland et al., May 1988, Seventh International Papillomavirus Workshop, Abstract, p. 5, report the use of PCR to detect HPV16 in oral and cervical biopsies. In addition, Campione-Piccardo et al., May 1988, Seventh International Papillomavirus Workshop, Abstract, p. 19, report the use of a mixture of primers for the specific amplification by PCR of HPV sequences regardless of type. A number of other researchers disclosed the use of PCR to amplify and detect HPV sequences at the Seventh International Papillomavirus Workshop.

Instruments for performing automated PCR are described in pending U.S. patent application Ser. No. 899,061, filed Aug. 22, 1986, which is a continuation-in-part of Ser. No. 833,368, filed Feb. 25, 1986, now abandoned. The preferred polymerase enzyme for use with PCR is Taq polymerase, isolated from Thermus aquaticus. Methods for purifying Taq polymerase and for the recombinant expression of the enzyme are disclosed in Ser. No. 143,441, filed Jan. 12, 1988, now abandoned, which is a continuation-in-part of Ser. No. 063,509, filed Jun. 17, 1987, which issued as U.S. Pat. No. 4,889,818, which is a continuation-in-part of Ser. No. 899,241, filed Aug. 22, 1986, now abandoned. The disclosures of these U.S. patents and patent applications are incorporated herein by reference.

Despite the use of PCR to amplify and detect HPV sequences, there still remains a need for a simple and rapid method for both detecting and typing HPV in a biological sample. The person invention provides a method that meets that need.

SUMMARY OF THE INVENTION

The present invention provides a method for detecting and typing HPV in a sample. The method comprises amplifying a sequence of HPV DNA present in the sample, determining if amplification has occurred, and then hybridizing an HPV type-specific probe to the amplified DNA. The invention also provides novel primers and probes for use in the method.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for detecting HPV in a sample, and typing the HPV if present, the method comprising:

(a) treating the sample with consensus HPV primers, an agent for polymerization, and deoxynucleoside 5'-triphosphates under conditions such that an extension product of a consensus primer can be synthesized, wherein said consensus primers are a mixture of oligonucleotides that comprises at least a pair of primers sufficiently complementary to separate single strands of HPV DNA to hybridize thereto so that the extension product synthesized from one member of said pair, when separated from its complementary strand, can serve as a template for synthesis of the extension product of the other member of said pair;

(b) separating the primer extension products, if present, from the templates on which the extension products were synthesized to form single-stranded molecules;

(c) treating the single-stranded molecules generated in step (b), if any, with the consensus primers of step (a) under conditions such that a primer extension product is synthesized using each of the single-stranded molecules produced in step (b) as a template;

(d) repeating steps (b) and (c) at least once;

(e) determining if amplification has occurred; and if amplification has occurred,

(f) hybridizing an HPV type-specific DNA probe to said amplified DNA; and

(g) determining if hybridization has occurred.

The first step in the method of the invention requires the use of consensus primer pairs that will amplify a discrete region of DNA from HPV DNA present in a sample. These consensus primer pairs are oligonucleotides, and the consensus primers are mixtures of primer pairs. The mixtures used in the method assure that, regardless of the type of HPV present in a sample, HPV DNA corresponding to the region between the "consensus" sequences will be amplified. The PCR products generated from the consensus primers, if HPV is present in the sample, are then analyzed by hybridization with type-specific probes to determine the HPV types present.

Amplification of DNA by the polymerase chain reaction (PCR) is disclosed in U.S. Pat. Nos. 4,683,202 and 4,683,195 and in the related applications noted and incorporated by reference in the Description of Related Disclosures, above. PCR amplification of DNA involves repeated cycles of heat-denaturing the DNA, annealing two oligonucleotide primers to sequences that flank the DNA segment to be amplified, and extending the annealed primers with DNA polymerase. The primers hybridize to opposite strands of the target sequence and are oriented so DNA synthesis by the polymerase proceeds across the region between the primers, effectively doubling the amount of that DNA segment. Moreover, because the extension products are also complementary to and capable of binding primers, each successive cycle essentially doubles the amount of DNA synthesized in the previous cycle. This results in the exponential accumulation of the specific target fragment, at a rate of approximately 2 n per cycle, where n is the number of cycles.

The choice of primers for use in PCR determines the specificity of the amplification reaction. In the amplification steps of the method of the present invention, "consensus" primers are used that will amplify HPV sequences present in a sample, regardless of type. The consensus primers of the invention can include degenerate primers, mixtures of the oligonucleotides synthesized so that any one of several nucleotides can be incorporated into a primer at a selected position during synthesis. The consensus primers are sufficiently complementary to all types of HPVs to amplify a DNA sequence of any HPV present in the sample. The consensus primers are also designed to amplify a region of DNA that contains sequences that are specific to each major viral type, so the amplified DNA can therefore be used to type the HPV present in the sample.

The invention, although applicable to any HPV, is exemplified below with reference to genital HPV strains. Furthermore, the primers are probes of the invention can be targeted to areas of the HPV genome other than those described below, provided that the particular area targeted can be amplified using consensus primers and the amplified DNA can be typed using type-specific probes.

The first step of the method of the present invention involves the amplification of an HPV sequence, if that sequence is present in a sample, by PCR using consensus primers. Illustrative consensus primers of the invention are referred to by the region of the HPV genome the primers are used to amplify. The HPV genome is circular. The genome of genital HPV is oriented as follows: E6, E7, E1, E2, E4, E5b, L2, L1, URR. "E" and "L" designations indicate open reading frames, but many of the open reading frames overlap. For instance, E4 is totally contained within the E2 open reading frame. URR is the transcriptional regulatory region. Primers can be used to amplify a sequence that spans one or more regions of the HPV genome.

For instances, the L1/E6 consensus primer combinations of the invention are designed to amplify a sequence of DNA from any genital HPV. The amplified sequence extends from L1 across the URR and into E6 and thus contains portions of the L1 and E6 regions with the URR region sandwiched in between the L1 and E6 regions. Thus, the consensus primer pairs consist of a first primer specific for a sequence in the L1 region and a second primer specific for a sequence in the E6 region. As shown in Table 1, below the first L1-specific primer can be either FS10, FS17, or MY01, while the second, E6-specific primer is at least a 1:1 mixture of J515 and J516, although the mixture can also contain more JS15 than JS16. Table 1 also depicts the sequence each primer and the corresponding sequence (and nucleotide position of that sequence) as it occurs in the genomes of several well-known genital HPVs (Types 6, 11, 16, 18, and 33). A dash in a sequence indicates that the genomic sequence is identical to the primer sequence. Nucleotides are abbreviated as follows:

    ______________________________________                                         Symbol Meaning      Origin                                                     ______________________________________                                         G      G            Guanine                                                    A      A            Adenine                                                    T      T            Thymine                                                    C      C            Cytosine                                                   R      G or A       puRine                                                     Y      T or C       pYrimidine                                                 M      A or C       aMino                                                      K      G or T       Keto                                                       S      G or C       Strong interaction (3 H bonds)                             W      A or T       Weak interaction (2 H bonds)                               H      A or C or T  not-G, H follows G in the alphabet                         B      G or T or C  not-A, B follows A                                         V      G or C or A  not-T (not-U), V follows U                                 D      G or A or T  not-C, D follows C                                         N      G or A or T or C                                                                            aNy                                                        ______________________________________                                    

                                      TABLE 1                                      __________________________________________________________________________     L1/E6 Consensus Primers and Amplification Products                             __________________________________________________________________________     L1 Consensus Positive Strand Primers                                           FS10 25 mer                                                                              5' C T G T G G T A G A T A C C A C A C G C A G T A C                 HPV06                                                                               6770    --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      --                                                                             --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               --                                                                               -- --               HPV11                                                                               6756    --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      --                                                                             --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               --                                                                               -- --               HPV16                                                                               6633    --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --      --                                                                             --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               --                                                                               -- --               HPV18                                                                               6587    --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      T                                                                              --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               --                                                                               -- --               HPV33                                                                               6587    --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      T                                                                              --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               --                                                                               -- --               FS17 20 mer                                                                              5' G A T C A G T T T C C Y Y T K G       G                                                                              A                                                                              C                                                                              G                           MY01 20 mer                                                                              5' G A T C A G T W T C C Y Y T K G       G                                                                              A                                                                              C                                                                              G                           HPV06                                                                               7151    --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               --                                                                               --                                                                               T T --                                                                               G --      --                                                                             --                                                                             --                                                                             --                          HPV11                                                                               7136    --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               C C --                                                                               T --      --                                                                             --                                                                             --                                                                             --                          HPV16                                                                               7015    --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               T T --                                                                               A --      --                                                                             --                                                                             --                                                                             --                          HPV18                                                                               6993    --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               A --                                                                               --                                                                               --                                                                               C C --                                                                               T --      --                                                                             --                                                                             --                                                                             --                          HPV33                                                                               6968    --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               T T --                                                                               G --      --                                                                             --                                                                             --                                                                             --                          URR/E6 Consensus Negative Strand Primer                                        JS15 18 mer                                                                              5' C C  G                                                                               T T T T C G G T   T S A A C C G                             HPV06                                                                                60     --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --  --                                                                               G --                                                                               --      --                                                                             --                                                                             --                          HPV11                                                                                60     --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --  --                                                                               G --                                                                               --      --                                                                             --                                                                             --                          HPV16                                                                                60     --                                                                               --                                                                               --                                                                               G --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --  --                                                                               C --                                                                               --      --                                                                             --                                                                             --                          HPV33                                                                                64     --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --  --                                                                               G --                                                                               --      --                                                                             --                                                                             --                          JS16 19 mer                                                                              5' C C G T T T T C G G T C C C G A       C                                                                              C                                                                              G                           HPV18                                                                                68     --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      --                                                                             --                                                                             --                                       Predicted Sizes Of PCR Products                                   + PRIMER     FS10      FS17 or MY01                                            - PRIMER     JS15 and JS16                                                                            JS15 and JS16                                           HPV06        1192 bp    822 bp                                                 HPV11        1235 bp    856 bp                                                 HPV16        1387 bp    958 bp                                                 HPV18        1367 bp    932 bp                                                 HPV33        1434 bp   1005 bp                                                 __________________________________________________________________________

As shown in Table 1, FS10 is a 25-mer that has 3 mismatches with HPV16 and 1 mismatch with HPV18 and HPV33, FS17 is a degenerate primer with 1 or 2 mismatches to different HPVs. MY01 is similar to FS17, contains 1 more degenerate base to decrease mismatches and to potentially cover a wider range of HPVs. JS15 is a degenerate 18-mer for the negative strand in E6 of HPVs 6, 11, 16, and 33, whereas JS16 is a 19-mer serving the same function for HPV18.

Once a sample has been treated with the L1/E6 primers shown above under conditions suitable for PCR, the method of the invention requires the determination of whether amplification has occurred. If amplification has occurred with the L1/E6 primers, HPV sequences are present in the sample. The use of an internal amplification control to assure the competency of a sample for PCR is within the scope of the invention and reduces the likelihood of false negative results. There are a variety of ways to determine whether amplification has occurred. A portion of the reaction mixture can be subjected to gel electrophoresis and the resulting gel stained with ethidium bromide and exposed to ultraviolet light to observe whether a product of the expected size is present. Labeled primers or deoxyribonucleotide 5'-triphosphates can be added to the PCR reaction mixture, and incorporation of the label into the amplified DNA is measured to determine if amplification occurred. Another method for determining if amplification has occurred is to test a portion of PCR reaction mixture for ability to hybridize to a labeled oligonucleotide probe designed to hybridize to only the amplified DNA. The probe must be a consensus probe so that amplified DNA from any HPV can be detected. For instance, amplification of HPV DNA using the L1/E6 consensus primers FS10, JS15, and JS16 can be detected using the L1/E6 consensus primer FS17 or MY01. Alternatively, the determination of amplification and identification of HPV type can be carried out in one step by testing a portion of the PCR reaction mixture for its ability to hybridize to one or more type-specific probes.

An important aspect of the present invention relates to the novel probes provided for use in the present methods. Whether these probes are consensus probes for determining if amplification has occurred or whether these probes are type-specific probes, the probes can be used in a variety of different hybridization formats. Although solution hybridization of a nucleic acid probe to a complementary target sequence is clearly within the scope of the present invention, commercialization of the invention will likely result in the use of immobility probes and thus a quasi "solid-phase" hybridization. In this format, the probe is covalently attached to a solid support and then the target sequences are then hybridized with the probe. A preferred method for immobilizing probes on solid supports is disclosed in U.S. patent application Ser. No. 197,000, filed May 20, 1988, incorporated herein by reference. In this method, the probes are attached to a solid support by virtue of long stretches of T residues; these T residues are added to the probe during synthesis of the probe on an automated synthesizer after the hybridizing sequence is synthesized. A variety of dyes and chromogens and corresponding labels are available for nucleic acid detection systems (see, e.g., U.S. patent application Ser. No. 136,166, filed Dec. 18, 1987, which describes the Kodak leuco-dye (TM) system).

The present invention has led and will continue to lead to the discovery of so many previously unknown (or at least uncharacterized) HPV types, however, that the embodiment of the invention in which consensus probes are used to determine if amplification has occurred will continue to decline in importance. This is because that with each new type of HPV discovered there comes a corresponding need to make the consensus probe more generic to ensure that the new type will be detected. To overcome this problem, the present invention provides a new type of consensus probe. This new type of probe essentially consists of a sequence DNA from one or more HPV viruses that comprises all or most of the DNA sequence that lies between the two sequences on the HPV genome corresponding to the primers used in the amplification. The consensus probe should not, however, comprise a sequence complementary to either primer used to amplify the HPV DNA in the sample. This new type of consensus probe has greater versatility in that it has more sequence available for hybridization than the other consensus probes of the invention. In addition, the consensus probe can be generated with PCR with primers that hydridize to sequences that lie inside the region of DNA defined by the primers used to amplify the HPV DNA sequences present in a sample.

The present invention provides a number of type-specific probes for use with the L1/E6 consensus primers of the invention. These probes are set forth in Table 2, below. Those skilled in the art will recognize that although the specific primers and probes of the invention exemplified herein have a defined number of nucleotide residues, one or more nucleotide residues may be added or deleted from a given primer or probe typically without great impact on the suitability of that primer or probe in the present methods.

                                      TABLE 2                                      __________________________________________________________________________     HPV Typing Probes For Use with L1/E6 Consensus Primers                         Specificity                                                                           Sequence             Size  Designation                                  __________________________________________________________________________     HPV6   5'CCAAACAGTAAGAGC    (15-mer)                                                                             FS18                                         HPV11  5'GGCTGTAGAGGGCTTAGAC                                                                               (19-mer)                                                                             FS19                                         HPV16  5'GGTTGAAGCTACAAAATGGGCC                                                                            (22-mer)                                                                             JS17                                         HPV18  5'GTAGCGCACCTGGACAGG (18-mer)                                                                             FS21                                         HPV33  5'CAGGTAGTGACTCAC    (15-mer)                                                                             FS22                                         __________________________________________________________________________

FS19 and JS17 can specifically detect HPV11 and HPV16, respectively. FS18 shows some hybridization with the HPV11 PCR product. UWGCG GAP program analysis comparing FS18 sequence and HPV11 sequence indicates a 73% homology of FS18 to HPV11 in the amplified region. The cross-hybridization could be minimized by increasing the stringency of washing. FS21 was specific for HPV18.

The L1/E6 primers disclosed above provide for the amplification of relatively large segments of HPV DNA. However, use of primers that result in shorter PCR products can have several advantages, including reduced extension and denaturation time and decreased denaturation temperature. The L1 consensus primers of the invention are illustrative of primers of the invention designed to amplify relatively small segments of the HPV genome to achieve such advantages. The L1 consensus primers produce a PCR product corresponding to sequences in the L1 open reading frame and are depicted in Table 3, below.

                                      TABLE 3                                      __________________________________________________________________________     L1 Consensus Primers and Amplification Products                                __________________________________________________________________________     L1 Consensus Positive Strand Primer                                            MY11                                                                               20 mer                                                                             5'                                                                               G C M C A G G G W C A T A A Y A A T G G                              HPV06                                                                              6722  --                                                                               --                                                                               C --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               C --      --                                                                             --                                                                             --                                                                             --                             HPV11                                                                              6707  --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               C --      --                                                                             --                                                                             --                                                                             --                             HPV16                                                                              6582  --                                                                               --                                                                               A --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               C --                                                                               --                                                                               C --                                                                               --                                                                               T --      --                                                                             --                                                                             --                                                                             --                             HPV18                                                                              6558  --                                                                               --                                                                               A --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               C --      --                                                                             --                                                                             --                                                                             --                             HPV33                                                                              6539  --                                                                               --                                                                               A --                                                                               --                                                                               A --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --      --                                                                             --                                                                             --                                                                             --                             L1 Consensus Negative Strand Primer                                            MY09                                                                               20 mer                                                                             5'                                                                               C G T C C M A R R G G A W A C T G A T C                              HPV06                                                                              7170  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               C --                                                                               A A --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               --      --                                                                             --                                                                             --                                                                             --                             HPV11                                                                              7155  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               G G --                                                                               --                                                                               --                                                                               A --                                                                               --                                                                               --      --                                                                             --                                                                             --                                                                             --                             HPV16                                                                              7033  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               A A --                                                                               --                                                                               --                                                                               A --                                                                               --                                                                               --      --                                                                             --                                                                             --                                                                             --                             HPV18                                                                              6712  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               G G --                                                                               --                                                                               --                                                                               T --                                                                               T --      --                                                                             --                                                                             --                                                                             --                             HPV33                                                                              6987  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               C --                                                                               A A --                                                                               --                                                                               --                                                                               A --                                                                               --                                                                               --      --                                                                             --                                                                             --                                                                             --                             Predicted sizes of PCR products from the                                       MY11 and MY09 L1 Consensus Primer Pair                                         HPV06 448 bp                                                                   HPV11 448 bp                                                                   HPV16 451 bp                                                                   HPV18 454 bp                                                                   HPV33 448 bp                                                                   __________________________________________________________________________

A preferred embodiment of the method of the present invention for genital HPVs comprises amplification of HPV sequences, if present in the sample, with the L1 consensus primers NY09 and MY11; determination of amplification by hybridization of a portion of the PCR reaction mixture with a generic genital HPV probe; and finally, type determination with type-specific probes. To determine if amplification of HPV DNA sequences has occurred in a sample that has been treated with the L1 consensus primers of the invention, a portion of the PCR reaction mixture can be hybridized with L1 consensus probes, depicted in Table 4.

                                      TABLE 4                                      __________________________________________________________________________     L1 Consensus Probes                                                            FS10     5'  C T G T G G T A G A T A C C A C A C G C A G T A C                 MY18     5'  --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --      --                                                                             --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               --                                                                               -- --               MY19     5'  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      T                                                                              --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               --                                                                               -- --               Sequence of HPV types in Region of Consensus Probe                             HPV6 6771                                                                               5'  C T G T G G  T                                                                               A G A T A C C A C A C G C A G T A C                 HPV11                                                                               6756                                                                               5'  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      --                                                                             --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               --                                                                               -- --               HPV16                                                                               6631                                                                               5'  --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --      --                                                                             --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               --                                                                               -- --               HPV18                                                                               6607                                                                               5'  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      T                                                                              --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               --                                                                               -- --               HPV33                                                                               6588                                                                               5'  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      T                                                                              --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               --                                                                               -- --               Isolate                                                                              38 5'  --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               G --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --      C                                                                              A                                                                              --                                                                             A --                                                                               --                                                                               C -- --               Isolate                                                                              88 5'  --                                                                               A --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --      T                                                                              --                                                                             --                                                                             --                                                                               --                                                                               --                                                                               C -- --               __________________________________________________________________________

When a portion of the PCR reaction mixture contains DNA that hybridizes to a probe contained in the L1 consensus probe mixture, the sample contains HPV DNA. There are a number of ways to determine whether a probe has hybridized to a DNA sequence contained in a sample. Typically, the probe is labeled in a detectable manner, the target DNA (i.e., the amplified DNA in the PCR reaction buffer) is bound to a solid support, and determination of whether hybridization has occurred simply involves determining whether the label is present on the solid support. This procedure can be varied, however, and works just as well when the target is labeled and the probe is bound to the solid support. The disclosure of U.S. patent application Ser. No. 197,000, filed May 20, 1988, incorporated herein by reference, describes preferred methods for using immobilized sequence-specific probes. Many methods for labelling nucleic acids, whether probe or target, are known in the art and are suitable for purposes of the present invention. In the laboratory work involved in reducing the present invention to practice, probes were typically labeled with radioactive phosphorous ³² P, by treating the probes with polynucleotide kinase in the presence of radiolabelled ATP. For commercial purposes, however, other non-radioactive labeling systems may be preferred, i.e., horseradish peroxidase-avidin-biotin systems.

Whatever the labeling system used, once a determination has been made that the L1 consensus probe has hybridized to amplified DNA present in the sample, the amplified DNA is typed to determine the HPV types present in the sample. Practice of the present invention has led to the discovery of many previously uncharacterized HPV types. For example, three clinical samples examined by the present method contained five different HPV types with sequences markedly different than the published sequences for HPVs. These new sequences are an important aspect of the present invention, as are the probes that will hybridize to these sequences in a type-specific fashion. These new sequences are depicted below. Degenerate nucleotides are as defined above and correspond to the degenerate nucleotides in the primers used to amplify the region or to variation within the type.

    __________________________________________________________________________     The DNA Sequence of the L1 Amplified Regions of HPV Isolates                   36A, 36B, 88, 238A, and 238B                                                   __________________________________________________________________________     Isolate 36A                                                                     1 GCMCAGGGWC                                                                               ATAAYAATGG                                                                               TATATGTTGG                                                                               CACAATCAAT                                                                               TGTTTTTAAC                           51                                                                               AGTTGTAGAT                                                                               ACTACTCGCA                                                                               GCACCAATCT                                                                               YTCTGTGTGT                                                                               GCTTCTACTA                          101                                                                               CTTCTCCTAT                                                                               TCCTAATGAA                                                                               TACACACCTA                                                                               CCAGTTTTAA                                                                               AGAATATGCC                          151                                                                               AGACATGTGG                                                                               RGGAATTTGA                                                                               TTTGCAGTTT                                                                               ATAYTTCAAC                                                                               TGTGTAAAAT                          201                                                                               AACWTTAACT                                                                               ACAGAGGTAA                                                                               TGTCATACAT                                                                               TCATAATATG                                                                               AATACCACTA                          251                                                                               TTTTGGAGGA                                                                               TTGGAATTTT                                                                               GGTRTTACAC                                                                               CACCTCCTAC                                                                               TGCTARTTTA                          301                                                                               GTTGACACAT                                                                               ACCGTTTTGT                                                                               TCAATCTGCT                                                                               GCTGTAACTT                                                                               GTCAAAAGGA                          351                                                                               CACCGCACCG                                                                               CCAGTTAAAC                                                                               AGGACCCTTA                                                                               TGACAAACTA                                                                               AAGTTTTGGA                          401                                                                               CTGTAAATCT                                                                               TAAGGAAAGG                                                                               TTTTCTGCAG                                                                               ATCTTGATCA                                                                               GTWTCCYYTK                          451                                                                               GGACG                                                                       Isolate 36B                                                                     1 GCMCAGGGWC                                                                               ATAAYAATGG                                                                               TATATGTTGG                                                                               GGAAATCAGC                                                                               TATTTTTAAC                           51                                                                               TGTGGTTGAT                                                                               ACTACCCGTA                                                                               GTACTAACAT                                                                               GACTTTGTGY                                                                               GCCACTGCAA                          101                                                                               CATCTGGTGA                                                                               TACATATACA                                                                               GCTGCTAATT                                                                               TTAAGGAATA                                                                               TTTAAGACAT                          151                                                                               GCTGAAGAAT                                                                               ATGATGTGCA                                                                               ATTTATATTT                                                                               CAATTGTGTR                                                                               AAATAACATT                          201                                                                               AACTGTTGAA                                                                               GTTATGTCAT                                                                               ATATACACAA                                                                               TATGAATCCT                                                                               AACATATTAG                          251                                                                               AGGAGTGGAA                                                                               TGTTGGTGTT                                                                               GCACCACCAC                                                                               CTTCAGGAAC                                                                               TTTAGAAGAT                          301                                                                               AGTTATAGGT                                                                               ATGTACAATC                                                                               AGAAGCTATT                                                                               CGCTGTCAGG                                                                               CTAAGGTAAC                          351                                                                               AACGCCAGAA                                                                               AAAAAGGATC                                                                               CTTATTCAGA                                                                               CTTTTCCTTT                                                                               TGGGAGGTAA                          401                                                                               ATTTATCTGA                                                                               AAAGTTTTCT                                                                               ACTGATTTAG                                                                               GATCAGTWTC                                                                               CYYTKGGACG                          Isolate 88                                                                      1 GCMCAGGGWC                                                                               ATAAYAATGG                                                                               CATATGCTGG                                                                               GGTAATCAGG                                                                               TATTTGTTAC                           51                                                                               TGTTGTGGAT                                                                               ACTACCAGAA                                                                               GCACCAACAT                                                                               GACTATTAAT                                                                               GCAGCTAAAA                          101                                                                               GCACATTARC                                                                               TAAATATGAT                                                                               GCCCGTGAAA                                                                               TCAATCAATA                                                                               CCTTCGCCAT                          151                                                                               GTGGAGGAAT                                                                               ATGAACTACA                                                                               GTTTGTGTTT                                                                               CAACTTTGTA                                                                               AAATAACCTT                          201                                                                               AACTGCAGAR                                                                               GTTATGGCAT                                                                               ATTTGCATAA                                                                               TATGAATAAT                                                                               ACTTTATTRG                          251                                                                               ACGATTGGAA                                                                               TATTGGCTTA                                                                               TCCCCACCAG                                                                               TTGCAACTAG                                                                               CTTAGAGGAT                          301                                                                               AAATATAGGT                                                                               ATATTAAAAG                                                                               CACAGCTRTT                                                                               ACAYGTCAGA                                                                               GGGAACAGCC                          351                                                                               CCCTGCAGAA                                                                               AAGCAGGATC                                                                               CCCTGGCTAA                                                                               ATATAAGTTT                                                                               TGGGAAGTTA                          401                                                                               ATTTACAGGA                                                                               CAGCTTTTCT                                                                               GCAGACCTGG                                                                               GATCAGTWTC                                                                               CYYTKGGACG                          Isolate 238A                                                                    1 GCMCAGGGWC                                                                               ATAAYAATGG                                                                               TATTTGTTGG                                                                               CATAATCART                                                                               TATTTTTAAC                           51                                                                               TGTTGTAGAT                                                                               ACTACTAGAA                                                                               GCACTAATTT                                                                               TTCTGTATGT                                                                               GTAGGTACAC                          101                                                                               AGGCTAGTAG                                                                               CTCTACTACA                                                                               ACGTATGCCA                                                                               ACTCTAATTT                                                                               TAAGGAATAT                          151                                                                               TTAAGACATG                                                                               CAGAAGAGTT                                                                               TGATTTACAG                                                                               TTTGTTYTTC                                                                               AGTTATGTAA                          201                                                                               AATTAGTTTA                                                                               ACTACTGAGG                                                                               TAATGACATA                                                                               TATACATTCT                                                                               ATGAATTCTA                          251                                                                               CTATATTGGA                                                                               AGAGTGGAAT                                                                               TTTGGTCTTA                                                                               CCCCACCACC                                                                               GTCAGGTACT                          301                                                                               TTAGAGGAAA                                                                               CATATAGATA                                                                               TGTAACATCA                                                                               CAKGCTATTA                                                                               GTTGCCAACG                          351                                                                               TCCTCAACCT                                                                               CCTAAAGAAA                                                                               CAGAGGACCC                                                                               ATATGCCAAG                                                                               CTATCCTTTT                          401                                                                               GGGATGTAGA                                                                               TCTTAAGGAA                                                                               AAGTTTTCTG                                                                               CAGAATTAGA                                                                               TCAGTWTCCY                          451                                                                               YTKGGACG                                                                    Isolate 238B                                                                    1 GCMCAGGGWC                                                                               ATAAYAATGG                                                                               TATTTGTTGG                                                                               GGCAATCAGT                                                                               TATTTGTTAC                           51                                                                               TGTGGTAGAT                                                                               ACCACACGTA                                                                               GTACCAATAT                                                                               GTCTGTGTGT                                                                               GCTGCAATTG                          101                                                                               CAAACAGTGA                                                                               TACTACATTT                                                                               AAAAGTAGTA                                                                               ATTTTAAAGA                                                                               GTATTTAAGA                          151                                                                               CATGGTGAGG                                                                               AATTTGATTT                                                                               ACRATTTATA                                                                               TTTCAGTTAT                                                                               GCAAAATAAC                          201                                                                               ATTATCTGCA                                                                               GACATAATGA                                                                               CATATATTCA                                                                               CAGTATGAAT                                                                               CCTGCTATTT                          251                                                                               TGGAAGATTG                                                                               GAATTTTGGA                                                                               TTGACCACAC                                                                               CTCCCTCAGG                                                                               TTCTTTAGAG                          301                                                                               GATACCTATA                                                                               GGTTTGTAAC                                                                               CTCACAGGCC                                                                               ATTACATGTC                                                                               AAAAARCTGC                          351                                                                               CCCCCAAAAG                                                                               CCCAAGGAAG                                                                               ATCCATTTAA                                                                               AGATTATGTA                                                                               TTTTGGGAGG                          401                                                                               TTAATTTAAA                                                                               AGAAAAGTTT                                                                               TCTGCAGATT                                                                               TAGATCAGTW                                                                               TCCYYTKGGA                          451                                                                               CG                                                                          __________________________________________________________________________

Those skilled in the art will recognize that with the above sequence information, primers and probes for amplifying and detecting these new HPV isolates can be readily obtained. In addition, these sequences enable one to isolate the entire virus from samples containing the virus. Isolate 238B is believed to correspond to the HPV31 type described in the literature. The discovery of these new HPV isolates led to the creation of new L1 consensus probes for use in conjunction with FS10, MY18, and MY19. These new L1 consensus probes are depicted below under the FS10 probe sequence to demonstrate the similarity to the FS10 sequence.

    __________________________________________________________________________     FS10                                                                               5'                                                                               C T G T G G T A G A T A C C A C A C G C A G T A C                        MY66  --                                                                               A --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               T       --                                                                             --                                                                             --                                                                             --                                                                               --                                                                               C --                                                                               --                       MY55  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               C       --                                                                             --                                                                             T                                                                              --                                                                               --                                                                               --                                                                               --                                                                               --                       MY39  --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               G --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               C       A                                                                              --                                                                             A                                                                              --                                                                               --                                                                               C --                                                                               --                       MY56  --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T --                                                                               --                                                                               T       A                                                                              --                                                                             A                                                                              --                                                                               --                                                                               C --                                                                               --                       MY57  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      --                                                                             --                                                                             T                                                                              --                                                                               --                                                                               --                                                                               --                                                                               --                       __________________________________________________________________________

As noted above, the diversity of HPV types may mandate the use of longer consensus probes that contain almost all of the amplified sequence except that portion corresponding to the primers used in the amplification step of the present method. This diversity in HPV types also demonstrates the need for the type-specific probes provided by the present invention.

The present invention provides a number of probes for typing amplified DNA produced from L1 consensus primers. These probes are depicted below in Table 5.

                                      TABLE 5                                      __________________________________________________________________________     HPV Typing Probes For Use with L1 Consensus Primers                                                              Genome                                       Probe Specificity                                                                           Sequence             Position                                     __________________________________________________________________________     MY12  HPV6   5'CATCCGTAACTACATCTTCCA                                                                             6813-6833                                    MY13  HPV11  5'TCTGTGTCTAAATCTGCTACA                                                                             6800-6820                                    MY14  HPV16  5'CATACACCTCCAGCACCTAA                                                                              6926-6945                                    WD74  HPV18  5'GGATGCTGCACCGGCTGA 6905-6922                                    MY16  HPV33  5'CACACAAGTAACTAGTGACAG                                                                             6628-6648                                    MY58  HPV16  5'TTGTAACCCAGGCAATTGCT                                                                              6897                                         MY59  HPV33  5'AAAAACAGTACCTCCAAAGGA                                                                             6877                                         MY60  HPV18  5'CAGTCTCCTGTACCTGGG 6657                                         MY61  HPV11  5'CACACCTGAAAAAGAAAAACAG                                                                            7051                                         MY62  HPV6   5'CTCCTGAAAAGGAAAAGCCA                                                                              7068                                         MY63  HPV6   5'TGGCTTTTCCTTTTCAGGAG                                                                              7068                                         MY64  HPV33  5'TCCTTTGGAGGTACTGTTTTT                                                                             6877                                         MY65  HPV11  5'CTGTTTTTCTTTTTCAGGTGTG                                                                            7051                                         WD126 HPV31  5'CCAAAAGCCCAAGGAAGATC                                                                              not available                                WD127 HPV31  5'CAAAAGCCCAAGGAAGATC                                                                               not available                                WD128 HPV31  5'TTGCAAACAGTGATACTACATT                                                                            not available                                __________________________________________________________________________

The present invention also provides consensus primers and HPV typing probes for detection of DNA sequences specific for the E6 region of genital HPVs. These probes are particularly preferred, because in some HPV-infected individuals, the HPV genome is partially deleted or rearranged such that only E6-related sequences are present. The E6 consensus primer pairs of the invention comprise primer pairs in which one primer is complementary to sequences near the border of the URR and E6 regions and the other primer is complementary to sequences in either the E7 region near the E6-E7 border (the E6 and E7 open reading frames overlap) or in the E6 region. These E6 consensus primers are depicted below in Table 6.

                                      TABLE 6                                      __________________________________________________________________________     E6 Consensus Primers                                                           __________________________________________________________________________     URR/E6 Consensus Positive Strand Primers                                       WD73   5'                                                                               C G G T T S A   A C C G A A A A C G G                                 WD72   5'                                                                               C G G T C G G G A C C G A A A A       C                                                                              G                                                                              G                               WD76   5'                                                                               C G G T T S A   A C C G A A A M       C                                                                              G                                                                              G                               WD77   5'                                                                               C G G T T C A   A C C G A A A M       C                                                                              G                                                                              G                               HPV06                                                                               43                                                                               5'                                                                               C G G T T C A   A C C G A A A A       C                                                                              G                                                                              G                               HPV11                                                                               43  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      --                                                                             --                                                                             --                              HPV16                                                                               43  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               G --  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               C --      --                                                                             --                                                                             --                              HPV18                                                                               43  --                                                                               --                                                                               --                                                                               --                                                                               C G G G --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      --                                                                             --                                                                             --                              HPV33                                                                               65  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      --                                                                             --                                                                             --                              The URR/E6 positive strand primers are used as mixtures: WD72                  and WD73; WD72 and WD76; and WD72 and WD77.                                    E7 Consensus Negative Strand Primer                                            WD70   5'                                                                               G C R C A G A T G G G R C A C A C                                     WD71   5'                                                                               G C A C A C C A C G G A C A C A       C                               HPV06                                                                              813                                                                               5'                                                                               G C G C A G A T G G G A C A C A       C                               HPV11                                                                              813  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      --                              HPV16                                                                              845  --                                                                               A A --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               G --                                                                               --                                                                               --                                                                               --      --                              HPV18                                                                              894  --                                                                               --                                                                               A --                                                                               --                                                                               C C A C --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      --                              HPV33                                                                              856  --                                                                               --                                                                               A --                                                                               --                                                                               --                                                                               G --                                                                               A --                                                                               --                                                                               G --                                                                               --                                                                               --                                                                               --      --                              WD68   5'                                                                               C A C A C A A T D Y Y Y A G T G       T                                                                              G                                                                              C                                                                              C C                             WD69   5'                                                                               C A C A C A A A G G A C A G G G       T                                                                              G                                                                              T                                                                              T C                             HPV06                                                                              801                                                                               5'                                                                               C A C A C T A T G T T T A G T G       T                                                                              T                                                                              C                                                                              C C                             HPV11                                                                              801  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               --                                                                               A --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --      --                                                                             G                                                                              --                                                                             --                                                                               --                            HPV16                                                                              833  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               --                                                                               T C C --                                                                               --                                                                               --                                                                               --                                                                               --      --                                                                             G                                                                              --                                                                             --                                                                               --                            HPV18                                                                              882  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               A --                                                                               G A C --                                                                               --                                                                               G --      --                                                                             G                                                                              T                                                                              T --                            HPV33                                                                              844  --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               --                                                                               A --                                                                               --                                                                               C --                                                                               C --                                                                               --      --                                                                             G                                                                              --                                                                             --                                                                               --                            The E7 negative strand primers are used as mixtures: WD70 and                  WD71; and WD68 and WD69.                                                       E6 Consensus Negative Strand Primer                                            WD67     W G C A W A T G G A W W G C Y G T C T C                               WD66     A G C A T G C G G T A T A C T G       T                                                                              C                                                                              T                                                                              C                               HPV6                                                                               286  T G C A T A T G G A T A G C C G       C                                                                              C                                                                              T                                                                              C                               HPV11                                                                              286  --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               G --                                                                               --                                                                               --                                                                               A --                                                                               --                                                                               T T --      T                                                                              --                                                                             --                                                                             --                              HPV16                                                                              286  A --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T C --                                                                               --                                                                               --                                                                               A       T                                                                              --                                                                             --                                                                             --                              HPV18                                                                              292  A --                                                                               --                                                                               --                                                                               --                                                                               G C --                                                                               --                                                                               T A T A --                                                                               T --      T                                                                              --                                                                             --                                                                             --                              HPV33                                                                              390  --                                                                               C --                                                                               --                                                                               A --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T T --                                                                               --                                                                               C       T                                                                              --                                                                             --                                                                             --                              __________________________________________________________________________     Predicted Sizes of Products from E6 Consensus Primers                                               Product Size for HPV Type                                 URR/E6 Primer                                                                              E7 Primer                                                                               HPV6    HPV11                                                                              HPV16 HPV18                                                                              HPV33                               __________________________________________________________________________     WD72 and WD73                                                                              WD70 and WD71                                                                           770     770 802   844 791                                 WD72 and WD76                                                                              WD70 and WD71                                                                           770     770 802   844 791                                 WD72 and WD77                                                                              WD70 and WD71                                                                           770     770 802   844 791                                 WD72 and WD73                                                                              WD68 and WD69                                                                           758     758 790   832 779                                 WD72 and WD76                                                                              WD68 and WD69                                                                           758     758 790   832 779                                 WD72 and WD77                                                                              WD68 and WD69                                                                           758     758 790   832 779                                 WD72 and WD73                                                                              WD66 and WD67                                                                           243     243 242   242 225                                 WD72 and WD76                                                                              WD66 and WD67                                                                           243     243 242   242 225                                 WD72 and WD77                                                                              WD66 and WD67                                                                           243     243 242   242 225                                 __________________________________________________________________________

Those skilled in the art will recognize that the E6 consensus primers of the invention amplify a sequence that comprises a portion of E7 DNA. To determine if amplification has occurred when the E6 consensus primers are used in the method of the invention, an E6 consensus probe is provided. The E6 consensus probes can also be used as E6 consensus positive strand primers. When used as E6 consensus primers, the E6 consensus probes are used in the following combinations: WD65 and WD64; WD83 and WD64; and WD84 and WD64. The E6 consensus probes are depicted in Table 7.

                                      TABLE 7                                      __________________________________________________________________________     E6 Consensus Probes                                                            __________________________________________________________________________     WD65   M G A G A C R G C W W T C C A T W T G                                   WD83   M G A G A C R G S W W T C C A T W    T                                                                         G                                       WD84   M G A G A C R G V W W T C C A T W    T                                                                         G                                       WD64   A G A G A C A G T A T A C C G C A    T                                                                         G                                       HPV6                                                                               267                                                                               C G A G G C G G C T A T C C A T A    T                                                                         G                                       HPV11                                                                              267                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A --                                                                               A A --                                                                               --                                                                               T --                                                                               --                                                                               --                                                                               C --                                                                               T    --                                                                        --                                      HPV16                                                                              266                                                                               A --                                                                               --                                                                               --                                                                               A T --                                                                               --                                                                               G A --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --   --                                                                        --                                      HPV18                                                                              273                                                                               A --                                                                               --                                                                               --                                                                               A --                                                                               A --                                                                               T A T A --                                                                               --                                                                               G C --   --                                 HPV33                                                                              271                                                                               A --                                                                               --                                                                               --                                                                               A G --                                                                               --                                                                               A A --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               T    --                                                                        --                                      __________________________________________________________________________

To determine the type of the HPV present in a sample when the E6 consensus primers are used in the method of the invention, E6 type-specific probes are provided. These probes are depicted in Table 8.

                                      TABLE 8                                      __________________________________________________________________________     HPV Typing Probes for Use with E6 Consensus Primers                                                            Genome                                         Probe                                                                               Specificity                                                                           Sequence            Position                                       __________________________________________________________________________     WD78 HPV6   5' CGAAGTGGACGGACAAGAT                                                                             643                                            WD79 HPV11  5' CAAGGTGGACAAACAAGACG                                                                            643                                            WD80 HPV16  5' GAACACGTAGAGAAACCCAG                                                                            534                                            WD81 HPV18  5' CAACCGAGCACGACAGGA                                                                              530                                            WD82 HPV33  5' GAGGTCCCGACGTAGAGAA                                                                             534                                            __________________________________________________________________________

The present invention also provides primers that are complementary to sequences in the HPV E1 region. These E1 primers can be used to amplify only E1 region sequences or can be used in conjunction with E6/E7 primers to amplify sequences from E6, E7, E1, and combinations of these three regions. These E1 primers are shown below in Table 9.

                                      TABLE 9                                      __________________________________________________________________________     E1 POSITIVE STRAND PRIMERS                                                     TYP01                                                                              20 MER                                                                              A T G G C K G A Y C C T G M A G G T A C                               TYP02                                                                              20 MER                                                                              --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               G A T T C --                                                                               --                                                                               --     --                                                                      --     --                             TYP03                                                                              20 MER                                                                              --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               C C T T C --                                                                               --                                                                               --     --                                                                      --     --                             TYP04                                                                              20 MER                                                                              T G T A M W G G M T G G T T T T A      T                                                                       G      T                              TYP05                                                                              20 MER                                                                              --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               G A      G                                                                       --     --                             TYP06                                                                              20 MER                                                                              --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A T      G                                                                       --     --                             E1 NEGATIVE STRAND PRIMERS                                                     TYN01                                                                              20 MER                                                                              G T A C C T K C A G G R T C M G C C A T                               TYN02                                                                              20 MER                                                                              --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               G A A T C --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --     --                                                                      --     --                             TYN03                                                                              20 MER                                                                              --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               G A A G G --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --     --                                                                      --     --                             TYN04                                                                              20 MER                                                                              A C A T A A A A C C A K C C W K T      A                                                                       C      A                              TYN05                                                                              20 MER                                                                              --                                                                               --                                                                               C T C --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --     --                                                                      --     --                             TYN06                                                                              20 MER                                                                              --                                                                               --                                                                               C A T --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --     --                                                                      --     --                             TYN07                                                                              20 MER                                                                              T C C A C T T C A G W A T T G C C      A                                                                       T      A                              TYN08                                                                              20 MER                                                                              --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               Y A --                                                                               --                                                                               --     --                                                                      --     --                             __________________________________________________________________________

These E1 primers can be used in a variety of embodiments of the present invention. For instance, amplifications wholly within the E1 region can be performed using the primer pairs: (1) TYP01, TYP02, TYP03, and TYN07, TYN08; or (2) TYP04, TYP05, TYP06 and TYN07, TYN08. Note that TYP03 is similar to both TYP02 and TYP01 and can be omitted from the amplification. The E1 region is highly conserved among HPVs, however, and although typing of the sample is possible with an E1 amplification, typing is more readily accomplished when the E1 primers are used in conjunction with E6 or E7 primers, as follows. For instance, one can amplify the E6/E7 region using the following E1 and E6/E7 primer pairs: (1) WD72, WD76 and TYN01, TYN02, TYN03; (2) WD64, WD 65 and TYN01, TYN02, TYN03; (3) WD72, WD76 and TYN04, TYN05, TYN06; (4) WD64, WD65 and TYN04, TYN05, TYN06; (5) WD72, WD76 and TYN07, TYN08; and (6) WD64, WD65 and TYN07, TYN08. In these latter amplifications, the entire E7 region is amplified. Thus, these amplification products can be detected with the E7 consensus probes depicted below.

    __________________________________________________________________________     TYP09                                                                               5' G A G C A A T T A G W A G A C                                          TYP12   --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               --                                                                               A R Y --                                                                               --                                                                               --                                         __________________________________________________________________________

Those skilled in the art recognize that the specific primers and probes disclosed herein are merely illustrative of the invention. For instance, because the primers and probes of the invention are single-stranded DNA molecules, and because the target DNA (HPV DNA in a sample) is double-stranded DNA, useful primers and probes of the invention can be constructed merely by synthesizing primers and probes complementary to those specifically disclosed herein. The primers and probes of the invention can also be prepared to amplify and detect sequence variations within areas of HPV genomes other than those specifically exemplified herein.

Primers of the invention are generally 18 to 21 nucleotides in length and are designed to have a high degree of homology with HPV sequences. For instance, in the design of the genital HPV consensus primers of the invention, a high degree of homology with all five major genital HPVs (HPV types 6, 11, 16, 18, and 33) was required. For each region to be amplified, two regions of homology are required, one for negative-strand primers and another for positive-strand primers. To identify a homology region, viral sequences are compared. Once a homology region is identified, a consensus primer is designed. Degenerate bases can be used in the design to accommodate positions at which an individual virus varies in sequence from the homology sequence. As many degenerate positions are made as is necessary so that all viral sequences have fewer than three mismatches with the consensus primer. The degenerate positions are chosen so that the smallest number of degenerate bases accommodates the largest possible number of viral sequences.

If a particular viral sequence has a large number of mismatches with the consensus sequence, then a type-specific primer is made for that virus. The type-specific primer is mixed with the degenerate primer that was designed for other viruses to make the consensus primer. Any mismatches that are not accommodated by the degenerate positions in the primer should generally be located more than 3 bases from the 3' end of the primer. Likewise, any degenerate positions should be more than 3 bases from the 3' end of the primer.

Estimated minimum and maximum T_(m) s for a degenerate primer should be between 54 and 64 degrees C. T_(m) s are estimated by the non-empirical formula: each G or C contributes 4 degrees C. to the T_(m) ; each A or T contributes 2 degrees C. to the T_(m) ; and the T_(m) is the sum of the calculated values. Finally, primers should not be designed to span palindromes or repetitive sequences.

Consensus probes design is similar to consensus primer design, except that consensus probes generally do not contain as many mismatches as consensus primers. As a result, the T_(m) for a probe can be higher than the T_(m) for a primer. However, where a mismatch or degenerate position occurs with respect to the 3' end is not as critical for consensus probes as it is for consensus probes.

Type-specific probes are designed so that a given probe will generally have less than 75% similarity with sequences from HPV types distinct from that recognized by the probe. The type-specific probes are usually 18-20 nucleotides in length with estimated T_(m) s in the range of 58 to 64 degrees C.

Those skilled in the art also recognize from the present disclosure that the method of the present invention can be carried out in a variety of ways. The present method is applicable to any human papillomavirus and especially preferred for detecting and typing genital HPVs. The method can be used to detect isolate-to-isolate variation within a particular HPV type and can also be used to screen for significant changes in the HPVs present in a patient. In one embodiment of the invention, consensus primers to more than one region of HPV DNA will be used, ensuring that if any portion of the HPV genome has been deleted, other regions can still be detected. In a similar fashion, the typing of the amplified DNA can be done using a variety of type-specific probes that recognize different regions of the amplified DNA.

Those skilled in the art recognize that the present invention can also be used to detect HPV mRNA present in a sample. The expression of certain HPV mRNA species, particularly E6 and E7 mRNAs, may be indicative of the likelihood than an HPV infection will progress to carcinoma. To detect an HPV mRNA by the method of the present invention, the mRNA can be treated with reverse transcriptase in an appropriate reaction mixture to synthesize a cDNA molecule. The primer used in the reverse transcription reaction can be a consensus primer of the invention or can be a different oligonucleotide that hybridizes near the 3' end of the mRNA. This cDNA copy is then made into a double stranded DNA molecule, which can be detected and typed in accordance with the method of the present invention.

The consensus primers of the present invention can also be used to detect HPV types previously uncharacterized. For instance, HPV isolates 36 and 88, noted in Table 4, above, have never before been characterized. Thus, the consensus primers of the invention can be used to amplify DNA sequences of previously unknown HPV types. The amplified DNA can then be sequenced and the sequence data used to generate type-specific probes for use in the method of the present invention.

The examples provided below merely illustrate the invention and in no way limit the scope of the accompanying claims.

EXAMPLE 1 Preparation of Clinical Samples for Amplification by the Polymerase Chain Reaction

Cervical and vulvar swabs and penile scrapes typically contain 10³ -10⁵ cells. Cells were suspended in 2 mL of phosphate-buffered saline, pelleted by centrifugation, and the supernatant discarded. If cell suspensions were to be stored for a period of time before the test, antibiotics were generally added to the suspension (a commercially available antibiotic, such as 2X Fungi Bact Solution, is suitable for this purpose). The cell number was estimated, and if blood was present in the sample, the cells were suspended in 1 mL of TE buffer (10 mM Tris-HCl, pH=7.5, and 1 mM EDTA) to lyse the red blood cells and then repelleted. About 10² -10⁴ cells were used per PCR reaction. The cells were first suspended in 100 μL of Taq buffer containing detergents and proteinase K to achieve a solution composed of the cells in 50 mM KCl; 10 mM Tris-HCl pH=8.3; 2 mM MgCl₂ ; 100 μg/mL gelatin; 0.45% NP40™; 0.045% Tween 20™; and 60 μg/mL proteinase K. This mixture was incubated at 55 degrees C. in a water bath for about 1 hours, and then the proteinase K was heat-inactivated by incubating the mixture at 95 degrees C. for 10 minutes. The samples were then treated in accordance with a standard PCR protocol with the consensus primers of the invention to amplify any HPV sequences present. Aliquots containing 10² -10⁴ cells were used per 100 μL of PCR reaction mixture.

Another method for preparing a cell suspension for PCR involves suspending the cells in 100 μL of deionized water (or deionized water containing 0.45% Tween 20™ and 0.45% NP40™) and incubating the resulting suspension at 100 degrees C. for 10-15 minutes.

EXAMPLE 2 Extraction of Tissue from Paraffin

The method of the present invention will be used to determine whether tissue sections in paraffin contain HPV. To prepare such a tissue section, typically 5-10 microns in width, for use in the present method, the following procedure was employed.

The tissue section was extracted twice with 1 mL of xylene or an alkene such as octane to remove the paraffin. Each extraction was for about one-half hour. The tissue section was then rinsed twice with 100% ethanol to remove the extracting agent and dried in a rotary evaporator. The section was then suspended in Taq buffer with detergents and proteinase K and treated as described in Example 1.

After heat inactivation of the proteinase K, the suspension was centrifuged to pellet debris, and about 10-20 μL of the supernatant were used for each PCR reaction.

EXAMPLE 3 PCR Protocols

All PCR protocols were carried out using a Perkin-Elmer/Cetus Instruments Thermal Cycler instrument. A typical reaction mixture contained 50 pmoles of consensus positive-strand primer; 50 pmoles of consensus negative-strand primer; 2.5 Units of Taq polymerase; 10 μl of 10X PCR buffer (0.5M KCl; 100 mM Tris-HCl, pH=8.3; 2 mM MgCl₂ ; and 1 mg/mL gelatin); 0.2 mM of each dNTP; about 10 μL of a clinical or paraffin sample; and deionized water to 100 μL.

PCR reaction times for use with clincal samples were as follows. The machine was heated to 72 degrees C. before the samples were placed into the machine. The machine was then programmed to execute the following temperature changes; one minute at 72 degrees C.; thirty cycles of 25 seconds at 95 degrees C., 25 seconds at 55 degrees C. and 50 seconds at 72 degrees C.; 5 minutes at 72 degrees C.; and then hold at 15 degrees C.

For paraffin sections, the machine was programmed to execute temperature changes as follows: 1 minute at 72 degrees C.; forty cycles of 50 seconds at 95 degrees C., 50 seconds at 55 degrees C., and 2 minutes at 72 degrees C.; 5 minutes at 72 degrees C.; and then hold at 15 degrees C.

If the PCR product was expected to be longer than 600 bp, the machine was programmed to execute temperature changes as follows: 1 minute at 72 degrees C.; forty cycles of a 50 second ramp to 95 degrees C., 20 seconds at 95 degrees C., a 90 second ramp to 55 degrees C., 30 seconds at 55 degrees C., a 50 second ramp to 72 degrees C., and 3 minutes at 72 degrees C.; 5 minutes at 72 degrees C.; and then hold at 15 degrees C.

To determine by use of consensus probes if amplification had occurred, about 10 μL of the reaction mixture were added to 140 μL of deionized water and 50 μL of 4X denaturing solution (1.6M NaOH and 100 mM EDTA). About 100 μL of this denatured solution were spotted onto a positively-charged, nylon Genetrans membrane using a BioRad dot-blot apparatus. The resulting dot was rinsed once with 200 μL of 2XSSC. The membrane was then removed from the blotter, air-dried, and exposed to ultraviolet light (with the DNA facing the light) for five minutes.

The membrane was pre-hybridized at least 45 minutes at 68 degrees C. in a water bath. The pre-hybridization solution contained 2XSSC, 10X Denhardt's solution, and 0.5% SDS. Alternatively, the membranes can merely be rinsed with pre-hybridization solution. The pre-hybridization solution was decanted. About 7 to 10 mL of the pre-hybridization solution were added to 100 μL of 10 mg/mL salmon sperm DNA and to about 1×10⁶ Cherenkov counts per mL of consensus probe (0.2 pmole). This solution was added back to the membrane, and the consensus probe was allowed to hybridize at 55 degrees C. for at least 3 hours. After the hybridization, the hybridization mix was decanted. The membrane was placed in a tray containing a room temperature wash solution composed of 2XSSC and 0.1% SDS. The tray was placed in a 55 degrees C. air-shaker for 10 minutes. This wash procedure was repeated once, and then the membrane was rinsed with a solution of 2XSSC and 0.1% SDS heated to 55 to 60 degrees C. An alternative wash procedure involves the same methodology but wash solutions at different temperatures, i.e., a 55 degrees C. wash solution in the first two washes and a room temperature wash solution in the final rinse. The membrane was then air-dried and allowed to expose X-ray film (generally, two intensifying screens were used) from 7 to 48 hours.

To determine the HPV type of amplified DNA, PCR reaction mixtures were hybridized to type-specific probes as described above. The only significant difference in the procedure was that the final wash of the filter was done at a temperature very near the Tm of the particular type-specific probe. Filters hybridized to different type-specific probes were not washed together.

Other modifications of the embodiments of the invention described above that are obvious to those of ordinary skill in the areas of molecular biology, medical diagnostic technology, biochemistry, virology, genetics and related disciplines are intended to be within the scope of the accompanying claims. 

We claim:
 1. AN HPV L1 consensus probe selected from the group consisting of probes MY66, MY55, MY39, MY56, and MY57. 